Altered calcium signalling in platelets from bile-duct-ligated rats

Abstract

In the present study, we have analysed the mechanisms of Ca(2+) entry and release in platelets obtained from BDL (bile-duct-ligated) rats, 11-13 days and 4 weeks after surgery. Platelets were washed and loaded with fura-2, and [Ca(2+)](i) (cytosolic Ca(2+) concentration) was determined in cell suspensions by means of fluorescence spectroscopy. Basal [Ca(2+)](i) was similar in platelets from BDL rats compared with those from their respective controls, both in the absence and presence of extracellular Ca(2+). Platelet stimulation with thrombin in the absence and presence of extracellular Ca(2+) induced a rapid rise in [Ca(2+)](i) that was of greater magnitude in platelets from BDL rats than in controls. Ca(2+) storage was significantly elevated in platelets from BDL rats, as well as the activity of SERCA (sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase). Capacitative Ca(2+) entry, as evaluated by inhibition of SERCA with thapsigargin, was also altered in platelets from BDL rats, having lower rates of Ca(2+) entry. In conclusion, chronic BDL alters intracellular Ca(2+) homoeostasis in platelets, such that an enhanced Ca(2+) release is evoked by thrombin, which may be due to an increased amount of Ca(2+) stored in the intracellular organelles and secondary to an enhanced activity of SERCA. These alterations are already evident before cirrhosis has completely developed and occurs during the cholestasis phase.

Bile Acids Do Not Contribute to the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis

“…Animals weighing ~250 g were subjected to bile-duct ligation (BDL) and excision or sham operation (controls), as described previously (Ortíz et al, 1996; Nadal et al, 2002; Atucha et al, 2003, 2007). Normal rat chow and tap water were offered ad libitum .…”

“…Several mechanisms have been suggested (Bowen et al, 1988; Pihusch et al, 2002; Atucha et al, 2007; Witters et al, 2010; Tripodi et al, 2011), including an intrinsic change in platelet function, e.g., increased aggregation resulting from altered intracellular calcium (Ca 2+ ) homeostasis or decreased aggregation due to a storage pool deficiency, the effect of a plasmatic factor such as bile acids or bilirubin or because of the effect of release of ADP-degrading enzymes in the circulation. Regarding platelet activation, the necessary increase in cytoplasmic Ca 2+ levels, both by release from internal stores and entry of extracellular Ca 2+ , has been reported to be defective both in experimental models of liver cirrhosis and patients (Bandi et al, 1997; Atucha et al, 2007; Annie-Jeyachristy et al, 2008). In previous studies from our laboratory, a greater amount of stored Ca 2+ together with an increased activity of SERCA, were observed in platelets obtained from a rat model of biliary cirrhosis (Atucha et al, 2007).…”

“…Regarding platelet activation, the necessary increase in cytoplasmic Ca 2+ levels, both by release from internal stores and entry of extracellular Ca 2+ , has been reported to be defective both in experimental models of liver cirrhosis and patients (Bandi et al, 1997; Atucha et al, 2007; Annie-Jeyachristy et al, 2008). In previous studies from our laboratory, a greater amount of stored Ca 2+ together with an increased activity of SERCA, were observed in platelets obtained from a rat model of biliary cirrhosis (Atucha et al, 2007). …”

Defective Platelet Activation and Bleeding Complications upon Cholestasis in Mice

“…Altered Ca 2+ signaling was described in bile duct ligated rats [24]. Atucha and colleagues found increased Ca 2+ mobilization following thrombin stimulation.…”

“…Few studies suggest a hypercoagulable state in patients with cholestatic liver disease [22, 23]. Atucha and colleagues described increased Ca 2+ responses of platelets from bile duct ligated rats [24]. In contrast to these studies showing platelet hyperactivity, hypoaggregability has been proposed from others with reduced expression of P-selectin, defective aggregation and prolonged bleeding times [15, 18, 23, 25].…”